Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.     

Native PAGE eliminates the problem of PEG-SDS interaction in SDS-PAGE and provides an alternative to HPLC in characterization of protein PEGylation

PEGylation of proteins has become an increasingly important technology in recent years. However, determination and characterization of the PEGylation products are problematic especially for the reaction mixture containing various modified proteins, unreacted PEG, and unmodified protein. A comparative study was carried out with two HPLC methods and two electrophoresis methods for characterization of the reaction mixture in PEGylation of HSA with PEG 5000, 10000, and 20000. RP-HPLC fails to give the correct information about the reaction of PEG 20000. Size-exclusion HPLC (SE-HPLC) produced very poor resolution on the PEG 5000 reaction. SDS-PAGE can run multiple samples of all PEGylation but the bands were smeared or broadened probably due to the interaction between PEG and SDS. On the other hand, native PAGE eliminates the problem of PEG-SDS interaction and provides better resolutions for all samples. Various PEGylated products and unmodified protein migrate differentially in native PAGE under nondenatured conditions. The results demonstrated that native PAGE could be a good alternative to HPLC and SDS-PAGE for the analysis of PEG-protein conjugates especially for characterization of the PEGylation mixture.     

Purification and modification by polyethylene glycol of a new human basic fibroblast growth factor mutant-hbFGF(Ser25,87,92)

The mutant of human basic fibroblast growth factor (hbFGF), hbFGF(Ser25,87,92), which was constructed by replacing the cysteine residues at the positions of the 25th, the 87th and the 92nd with serine residues, was coupled to polyethylene glycol (PEG) with a molecular size of 20 kDa (20K) (PEG(20K)) to obtain hbFGF derivative, PEG(20K)-hbFGF(Ser25,87,92). The optimal modified reaction was conducted at 12 degrees C for 12h with the molar ratio of PEG(20K) to hbFGF(Ser25,87,92) of 30:1. The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the modification rate was up to 60%. The PEGylated product retained binding affinity to heparin and could be purified by heparin affinity chromatography. Compared to hbFGF mutant, purified PEG(20K)-hbFGF(Ser25,87,92) retained about 34% of mitogenic activity. Heat-stability assay indicated that the modified product was more stable than the native protein at the temperature of 37 degrees C.     

Membrane chromatography: Protein purification from E. coli lysate using newly designed and commercial anion-exchange stationary phases

This contribution describes the purification of anthrax protective antigen (PA) protein from Escherichia coli lysate using bind-and-elute chromatography with newly designed weak anion-exchange membranes. Protein separation performance of the new AEX membrane adsorber was compared with the commercial Sartobind^® D membrane adsorber and HiTrap™ DEAE FF resin column under preparative scale conditions. Dynamic protein binding capacities of all three stationary phases were determined using breakthrough curve analysis. The AEX membrane showed higher binding capacities than the Sartobind^® D membrane at equivalent volumetric throughput and higher capacities than the HiTrap™ DEAE FF resin column at 15 times higher volumetric throughput. Anion-exchange chromatography was performed using all three stationary phases to purify PA protein. Quantitative SDS-PAGE analysis of effluent fractions showed that the purity of PA protein was higher for membrane adsorbers than the HiTrap™ DEAE FF resin column and was the same for the new AEX membrane and Sartobind^® D membrane adsorbers. The effects of E. coli lysate load volume and volumetric flow rate on PA protein separation resolution using the membrane adsorbers were minor, and the peak elution profile remained un-changed even under conditions where >75% of the total protein dynamic binding capacity of the membranes had been utilized. PA protein peak resolution was higher using pH-gradient elution than with ionic strength gradient elution. Overall, the results clearly demonstrate that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher volumetric throughput.     

Trapping of Thiol Terminated Acrylate Polymers with Divinyl Sulfone to Generate Well-Defined Semi-Telechelic Michael Acceptor Polymers

Herein we report the synthesis of vinyl sulfone end functionalized PEGylated polymers by reversible addition-fragmentation chain transfer (RAFT) polymerization for conjugation to proteins. Poly(ethylene glycol) methyl ether acrylate (PEGA) was polymerized in the presence of 1-phenylethyl dithiobenzoate with 2,2'-azobis(2-methylpropionitrile) as the initiator to generate well-defined polyPEGAs with number-average molecular weights (M(n)) by gel permeation chromatography (GPC) of 6.7 kDa, 11.8 kDa and 16.1 kDa. Post-polymerization, the majority of polymer chains contained the dithioester functional group at the omega chain end, and the polydispersity indexes (PDI) of the polymers ranged from 1.08 to 1.24. The dithioester was subsequently reduced via aminolysis, and the resulting thiol was trapped with a divinyl sulfone in situ to produce semi-telechelic, vinyl sulfone polyPEGAs with efficiencies ranging between 85% and 99%. It was determined that the retention of vinyl sulfone was directly related to reaction time, with the maximum dithioester being transformed into a vinyl sulfone within 30 minutes. Longer reaction times resulted in slow decomposition of the vinyl sulfone end group. The resulting semi-telechelic vinyl sulfone polymers were then conjugated to a protein containing a free cysteine, bovine serum albumin (BSA). Gel electrophoresis demonstrated that the reaction was highly efficient and that conjugates of increasing size were readily prepared. After polymer attachment, the activity of the BSA was 92% of the unmodified biomolecule.     

Purification and analysis of mono‐PEGylated HSA by hydrophobic interaction membrane chromatography

We discuss the purification of mono‐PEGylated HSA by hydrophobic interaction membrane chromatography. The hydrophobicity difference between the different fractionated species was induced by the addition of a lyotropic salt that caused phase transition of PEG (hydrophilic under normal condition) to a mildly hydrophobic form. The HSA PEGylation reaction mixture was mixed with lyotropic salt and passed through a stack of hydrophilized polyvinylidene fluoride membrane discs. Unmodified HSA was obtained in the flow through, while the PEGylated forms of the protein bound to the membrane and could be eluted by reducing the salt concentration. Among the three major PEGylated forms of HSA present in the feed (i.e. mono–, di–, and tri–), mono‐PEGylated HSA was eluted first and could be resolved from the others. The purified material was analyzed by SDS‐PAGE, dynamic light scattering, and SEC combined with multi‐angle light scattering. All these analytical techniques indicated the presence of species that has a molar mass consistent with mono‐PEGylated HSA. A scaled‐down version of the membrane chromatographic methods could be used for the rapid and sensitive analysis of PEGylated proteins.     

Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media

The effect of PEGylation on cation exchange chromatography was studied with poly(ethylene glycol) of different chain lengths (5 kDa, 10 kDa and 30 kDa) using lysozyme as a model system. A stable binding via reduction of a Schiff base was formed during random PEGylation on lysine residues with methoxy-PEG-aldehyde. A purification method for PEGylated proteins using cation exchange chromatography was developed, and different isoforms of mono-PEGylated lysozyme were isolated. TSKgel SP-5PW and Toyopearl GigaCap S-650M showed the best performance of all tested cation exchange resins, and the separation of PEGylated lysozyme could be also scaled up to semi-preparative level. Size-exclusion chromatography, SDS-PAGE and MALDI-TOF mass spectrometry were used for analysis. Separated mono-PEGylated lysozyme of different sizes was used to determine dynamic binding capacities (DBC) and selectivity of cation exchange chromatography resins. An optimization of binding conditions resulted in a more than 20-fold increase of DBC for Toyopearl GigaCap S-650M with 30 kDa mono-PEGylated lysozyme.     

Microcalorimetric study of the adsorption of PEGylated lysozyme on a strong cation exchange resin

Adsorption of native as well as mono-, di-, and tri-PEGylated lysozyme on Toyopearl Gigacap S-650M in sodium phosphate buffer is studied by isothermal titration calorimetry and by independent adsorption equilibrium measurements at pH 6 and 25°C. The production and separation of PEGylated lysozyme is discussed. Two different PEG sizes are used (5 kDa and 10 kDa) which leads to six different forms of PEGylated lysozyme which were systematically studied. The sodium chloride concentration is varied according to the elution conditions in the production process. The specific enthalpy of adsorption Δh(p)(ads) is determined from the calorimetric and the adsorption equilibrium data. It was found to be exothermal and constant with increasing adsorber loading for native lysozyme. For all PEGylated forms there is an influence of the adsorber loading on Δh(p)(ads) which is found to become more important with increasing PEGylation degree (total molecular weight of conjugated PEG). At low loadings the adsorption of all PEGylated lysozyme forms is exothermal. With increasing loading the adsorption becomes less exothermal and for the species with higher PEGylation degree also endothermal effects are observed at higher loadings. A thermodynamic analysis is carried out by which the enthalpic and entropic contributions to the binding constants are quantified. The findings are discussed on a molecular level. The results provide insight into the adsorption mechanisms of polymer-modified proteins on chromatographic resins.     

Comparison of Anion-Exchange and Hydrophobic Interactions between Two New Silica-Based Long-Chain Alkylimidazolium Stationary Phases for LC

Two new silica-based long-chain alkylimidazolium stationary phases were prepared and characterized for their use in high-performance liquid chromatography. The stationary phases were both prepared by the reaction of chloropropyl silica with long-chain alkylimidazoles and were used to separate common inorganic anions. Hydrophobic interactions were also studied by the comparison of differential retention of various organic compounds. The alkyl chain length did not show an impact on the anion-exchange process but affected the hydrophobic interaction of the stationary phases.     

离子交换色谱法分离纯化鸡卵黄免疫球蛋白

建立了高效、经济、大规模获得鸡卵黄免疫球蛋白(IgY)的生产方法。在对传统的水稀释法改良的基础上,结合聚乙二醇沉淀与离子交换色谱进行IgY的分离纯化。结果显示,用8倍无菌水稀释蛋黄液,用0.1 mol/L HCl调节pH为5.2,在4 ℃下静置8 h,于5000×g力离心可得上清粗IgY液,经测定回收率可达93.47%。然后用6%聚乙二醇沉淀后经DEAE-Toyopearl 650 M离子交换纯化,最佳的纯化条件: 0.05 mol/L磷酸盐缓冲液(PBS, pH 7)平衡上样,0.075 mol/L PBS(pH 7)洗脱。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果显示所得的IgY的纯度为95.02%,活性保持率高达73.77%。本研究弥补了传统分离方法不能同时达到高纯度和高回收率的缺点,且可用于大规模生产。     

Affinity partitioning of plasmid DNA with a zinc finger protein

The affinity isolation of pre-purified plasmid DNA (pDNA) from model buffer solutions using native and poly(ethylene glycol) (PEG) derivatized zinc finger-GST (Glutathione-S-Transferase) fusion protein was examined in PEG-dextran (DEX) aqueous two-phase systems (ATPSs). In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected in the PEG phase of a PEG 600-DEX 40 ATPS. This represents a 1322-fold increase in the protein partition coefficient in comparison to the non-PEGylated protein (Kc = 0.013). In the presence of pDNA containing a specific oligonucleotide recognition sequence, the zinc finger moiety of the PEGylated fusion protein bound to the plasmid and steered the complex to the PEG-rich phase. An increase in the proportion of pDNA that partitioned to the PEG-rich phase was observed as the concentration of PEGylated fusion protein was increased. Partitioning of the bound complex occurred to such an extent that no DNA was detected by the picogreen assay in the dextran phase. It was also possible to partition pDNA using a non-PEGylated (native) zinc finger-GST fusion protein in a PEG 1000-DEX 500 ATPS. In this case the native ligand accumulated mainly in the PEG phase. These results indicate good prospects for the design of new plasmid DNA purification methods using fusion proteins as affinity ligands.     

Influence of surface modification on protein retention in ion-exchange chromatography: Evaluation using different retention models

A large number of different stationary phases for ion-exchange chromatography (IEC) from different manufacturers are available, which vary significantly in a number of chemical and physical properties. As a consequence, binding mechanisms may be different as well. In the work reported here, the retention data of model proteins (α-lactalbumin, β-lactoglobulin A, bovine serum albumin and alcohol dehydrogenase) were determined for three anion-exchange adsorbents based on synthetic copolymer beads with differences in the functional group chemistry. Fractogel EMD DEAE and Fractoprep DEAE consist of functional groups bound to the surface via “tentacles”, ToyopearlDEAE by a short linker. Three models which describe chromatographic retention were used to analyse the characteristic parameters of the protein/stationary-phase interactions. The number of electrostatic interaction between the stationary phase and the model proteins, the protein specific surface charge densities and the interacting surface of the proteins with the adsorptive layer of the chromatographic media depend on the surface modification as well as on the molecular mass of the model proteins. In general, protein retention of the model proteins on the weak anion exchangers was found to be greater if the stationary phase carries tentacles and protein mass is above 60 kDa.     

Purification and identification of PEGlated hemoglobin, a potential blood substitute, by chromatography and capillary electrophoresis

Summary Bovine hemoglobin (Hb) has been chemically modified, by reaction of its lysine residues with the active ester of poly(ethylene glycol) (PEG,M _w=5000), to produce a potential blood substitute for human therapy. Covalent attachment of PEG chain to the protein produced a heterogeneous mixture of Hb from the mixture. This paper describes the use of cation-exchange chromatography (IEC), in flow-through mode, and size-exclusion chromatography (SEC) for purification of the PEG-Hb mixture. The highly modified Hb flowed through the IEC column in the loading buffer without adsorption by the chromatographic medium. SEC was then used for further purification. These two steps were suitable for pilot-scale preparation or for analytical chromatography. The purified product was assessed by high-performance capillary electrophoresis (HPCE), which was also used to optimize the chromatographic parameters.     

Chromatographic analysis of tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle.

Tropomyosins from rabbit skeletal, chicken gizzard and earthworm muscle all exist as dimeric, ca. 100% alpha-helical coiled-coil species in benign media. Two major tropomyosin isoforms from each muscle source have been identified and can be conveniently designated alpha (fast) and beta (slow) based on electrophoretic mobility under denaturing conditions. The ratio of alpha to beta chains is ca. 3-4:1 for rabbit skeletal and ca. 1:1 for chicken gizzard and earthworm tropomyosins. Each chain from the former two muscle sources has been sequenced, thus providing a molecular basis for interpreting the in vivo population of homo- and hetero-dimers. The characteristics of each purified tropomyosin in weak-anion exchange, strong-cation exchange and reversed-phase high-performance liquid chromatography are described. Binding to and/or elution from the reversed-phase matrix results in dissociation into highly helical monomeric chains. This mode of chromatography separates the alpha and beta chains of earthworm and chicken gizzard tropomyosins, but not those of the rabbit protein. Both anion- and cation-exchange chromatography use mild (benign) elution conditions under which the native, in vivo dimer population should be preserved. Only the rabbit protein exhibited peak separation on the anion-exchange resin, with peak assignment corresponding to the known molecular organization of homo- and hetero-dimers. In strong cation-exchange analysis, all three tropomyosins exhibit a chromatographic transition near pH 6.5, possibly the result of histidine(s) titration. Collectively, the chromatographic data confirm the present understanding of the in vivo mixture of dimers for tropomyosin from rabbit skeletal and chicken gizzard. It is concluded that native earthworm tropomyosin exists predominantly as an alpha beta hetero-dimer.     

Chromatographic behavior of mPEG-papaya proteinases conjugates examined on ion-exchange and hydrophobic gel media

The four cysteine proteinases, papain, chymopapain, caricain, and endoproteinase Gly-C were isolated and purified as the catalytically competent species from the commercially available latex of the tropical treeCarica papaya L. Their free thiol function (cysteine-25), which is essential for activity, was protected in the form of a mixed disulfide containing a 5 kDa polyethylene glycol (PEG) chain. The second (nonessential) free thiol function (cysteine-117) of chymopapain was blocked similarly. Caricain was also derivatized through acylation of its amino functions by PEG chains (average: 15 moles of PEG per mole of enzyme). The Chromatographic behavior of these conjugates was examined on ion-exchange and hydrophobic gels and compared to the Chromatographic behavior of the unpegylated proteinases. The results indicated that charge-shielding effects by PEG chain(s), surrounding the different proteinases, plays(play) a key role in the course of separation of pegylated and unpegylated species by ion-exchange chromatography. Similarly, PEG chain(s) is(are) able to mask hydrophobic regions on the surface of the proteinases. However, the affinity showed by PEG itself for the hydrophobic ligands immobilized on the matrix is the preponderant factor determining the behavior of the PEG-proteinases conjugates on Fractogel TSKButyl-650.     

The preparation of bovine serum albumin surface-imprinted superparamagnetic polymer with the assistance of basic functional monomer and its application for protein separation

Currently, small proteins imprinting are more reported since large proteins molecular imprinting faces challenge due to their bulk size and complex structure. In this work, bovine serum albumin (BSA) surface-imprinted magnetic polymer was successfully synthesized based on atomic transfer radical polymerization (ATRP) method in the presence of common monomer (N-isopropylacrylamide) with the assistant of basic functional monomer (N-[3-(dimethylamino)propyl]-methacrylamide), which provides a achievable attempt for imprinting larger target proteins based on the ATPR with the mild reaction conditions. The BSA-imprinted polymer exhibited higher adsorption capacity and selectivity to BSA over the non-imprinted polymer. Competitive adsorption tests indicated the BSA-imprinted polymer had better selective adsorption and recognition properties to BSA in the mixture. The obtained BSA-imprinted polymer was applied to bovine serum, which also showed selectivity to BSA. In addition, a conventional aqueous two-phase solution of PEG/sulphate was used as elution for adsorbed BSA, which was compared with common NaCl elution.     

聚乙二醇修饰蛋白体系的SDS-聚丙烯酰胺凝胶电泳染色方法新探

比较了聚乙二醇修饰蛋白体系的SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)银染、考染、碘化钡染色3种染色方法;提出和比较了银染-碘化钡复染和考染-碘化钡复染2种复染方法.结果表明,银染-碘化钡复染的凝胶中,未修饰蛋白条带消失,PEG修饰蛋白条带保留,游离PEG条带显色;而考染-碘化钡复染的凝胶中,未修饰蛋白、修饰蛋白和游离的PEG条带可同时显色.两种复染方法中,PEG组分的检测限均达到了0.01μg.因此,对PEG修饰蛋白体系的SDS-PAGE可先用考染或银染后再用碘化钡复染,便可在同一块凝胶上先后或同时观察到未修饰蛋白、修饰蛋白和游离PEG的情况,简化了实验操作,方便了实验结果的比较分析.     

阴离子交换整体柱对蛋白质的分离与纯化

分别用乙二胺、二乙胺、三乙胺将自制的以甲基丙烯酸缩水甘油酯(GMA)为单体、乙二醇二甲基丙烯酸酯(EDMA)为交联剂的整体柱修饰为弱、强阴离子交换整体柱。考察了该整体柱的性能,选择出分离蛋白质(牛血清白蛋白、溶菌酶和谷胱甘肽)的最佳实验条件,并在最佳分离条件下考察了这些蛋白质在整体柱上的色谱行为和该整体柱对纤维素降解酶的分离纯化情况。实验结果表明,该整体柱性能良好,可以实现对纤维素降解酶的快速分离与纯化。同时,实验也证明采用梯度洗脱可以实现对某些蛋白质的分离纯化。     

Effects of large sample loads on column lifetime in preparative-scale liquid chromatography

In preparative-scale liquid chromatography of proteins, the use of high sample concentration and large sample mass may result in irreversible adsorption to the support surface. This can change the stationary phase characteristics, reduce the capacity, shorten the column lifetime and diminish the economic viability of a particular separation method. Column recycling and regeneration can influence the throughput (mass purified per time unit) and selectivity, and affect the reproducibility. The effects of large sample loads on column lifetime and performance were evaluated for three strong anion-exchange columns: (1) a silica support with a quaternized polyethyleneimine (PEI) coating, (2) a polymeric support with an adsorbed PEI coating which also was quaternized, and (3) a polymeric support with a proprietary quaternary amine stationary phase. The column capacity for proteins was measured by frontal chromatography and monitored as a function of cycle number. The column lifetime was determined by examining chromatographic properties subsequent to the frontal chromatography. The change in protein binding capacity was then compared to the change in nitrate binding capacity. The column performance was evaluated under analytical conditions by measuring the change in resolution of standard protein mixtures.     

Engineering of a two-step purification strategy for a panel of monoclonal immunoglobulin M directed against undifferentiated human embryonic stem cells

A two-step purification strategy comprising of polyethylene glycol (PEG) precipitation and anion-exchange chromatography was developed for a panel of monoclonal immunoglobulin M (IgM) (pI 5.5–7.7) produced from hybridoma cultures. PEG precipitation was optimized with regards to concentration, pH and mixing. For anion-exchange chromatography, different resins were screened of which Fractogel EMD, a polymer grafted porous resin had the highest capacity. Despite its significantly slower mass transfer, the binding capacity was still higher compared to a convection driven resin (monolith). This purification strategy was successfully demonstrated for all 9 IgMs in the panel. In small scale most antibodies could be purified to >95% purity with the exception of two which gave a lower final purity (46% and 85%). The yield was dependent on the different antibodies ranging from 28% to 84%. Further improvement of recovery and purity was obtained by the digestion of DNA present in the hybridoma supernatant using an endonuclease, benzonase. So far this strategy has been applied for the purification of up to 2 l hybridoma supernatants.